Redox-coupled proton transfer mechanism in nitrite reductase revealed by femtosecond crystallography

Significance Copper nitrite reductase (CuNiR) is involved in denitrification of the nitrogen cycle. Synchrotron X-rays rapidly reduce copper sites and decompose the substrate complex structure, which has made crystallographic studies of CuNiR difficult. Using femtosecond X-ray free electron lasers, we determined intact structures of CuNiR with and without nitrite. Based on the obtained structures, we proposed a redox-coupled proton switch model, which provides an explanation for proton-coupled electron transfer (PCET) in CuNiR. PCET is widely distributed through biogenic processes including respiratory and photosynthetic systems and is highly expected to be incorporated into bioinspired molecular devices. Our study also establishes the foundation for future studies on PCET in other systems. Proton-coupled electron transfer (PCET), a ubiquitous phenomenon in biological systems, plays an essential role in copper nitrite reductase (CuNiR), the key metalloenzyme in microbial denitrification of the global nitrogen cycle. Analyses of the nitrite reduction mechanism in CuNiR with conventional synchrotron radiation crystallography (SRX) have been faced with difficulties, because X-ray photoreduction changes the native structures of metal centers and the enzyme–substrate complex. Using serial femtosecond crystallography (SFX), we determined the intact structures of CuNiR in the resting state and the nitrite complex (NC) state at 2.03- and 1.60-Å resolution, respectively. Furthermore, the SRX NC structure representing a transient state in the catalytic cycle was determined at 1.30-Å resolution. Comparison between SRX and SFX structures revealed that photoreduction changes the coordination manner of the substrate and that catalytically important His255 can switch hydrogen bond partners between the backbone carbonyl oxygen of nearby Glu279 and the side-chain hydroxyl group of Thr280. These findings, which SRX has failed to uncover, propose a redox-coupled proton switch for PCET. This concept can explain how proton transfer to the substrate is involved in intramolecular electron transfer and why substrate binding accelerates PCET. Our study demonstrates the potential of SFX as a powerful tool to study redox processes in metalloenzymes.

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